Immune system regulation using novel phenylalkanoic acid derivative

ABSTRACT

A method of regulating the immune system of a mammal, which comprises administering to said mammal an immuno-regulatory agent, wherein said agent is selected from the group consisting of compounds of the formula (I):   &lt;IMAGE&gt; (I)  (wherein R represents hydrogen or a C1-C3 alkyl group) and pharmaceutically acceptable salts and esters thereof.

BACKGROUND TO THE INVENTION

The present invention relates to a new series of phenylalkanoic acidderivatives, which have been found to have anti-inflammatory andanalgesic activities and some of which have also been found to haveimmuno-regulatory activity. The invention also provides methods of usingthese compounds.

Mild analgesic and anti-inflammatory agents are amongst the mostcommonly used of drugs. Most such drugs in common use have side effectswhich may be distressing or even dangerous to a small percentage of thepopulation--even though the number of people so afflicted may bestatistically insignificant, it is better for such persons to employ adifferent analgesic or anti-inflammatory drug, whose side effects maynot be distressing or dangerous to them, rather than to continue withthe original drug. There is, therefore, a continuing need for newanalgesic and anti-inflammatory drugs, to broaden the choice availableto the consumer.

The known mild analgesic and anti-inflammatory agents fall into a numberof recognised chemical classes; one such class consists ofphenylalkanoic acid derivatives, which mainly vary in accordance withthe nature of the substituents on the phenyl group. A series ofcompounds within this class is disclosed, for example, in British PatentSpecification No. 2,113,214 and another such series is disclosed in U.S.Pat. No. 4,400,534.

BRIEF SUMMARY OF THE INVENTION

It is an object of the present invention to provide a series of newphenylalkanoic acid derivatives having good analgesic andanti-inflammatory activities.

The compounds of the present invention are those compounds of formula(I): ##STR2## (wherein R represents hydrogen or a C₁ -C₃ alkyl group)and pharmaceutically acceptable salts and esters thereof.

The invention also provides a method of treating a mammal byadministering thereto an analgesic and anti-inflammatory agent of thecompound (I).

The invention still further provides a method of regulating the immunesystem of a mammal by administering thereto an immuno-regulatory drug,wherein said immuno-regulatory drug is selected from the compounds ofthe invention.

DETAILED DESCRIPTION OF INVENTION

The compounds of the invention can exist in the form of geometricisomers, depending upon the relative positions of the substituents onthe cyclohexane ring and the present invention contemplates theindividual isolated isomers, as well as mixtures thereof, specifically,the cis-isomer, which may be represented by the formula (II): ##STR3##and the trans-isomer, which may be represented by the formula (III):##STR4## (wherein R is a defined above), as well as pharmaceuticallyacceptable salts and esters thereof.

Each of these geometric isomers also exists in the form of two opticalisomers, which may be separated from each other by conventionalresolution techniques or may be left unresolved.

Additionally, the presence of an asymmetric carbon atom adjacent thecarboxyl group in the compounds of the invention means that each of thecompounds represented by the above formulae can exist in the form ofoptical isomers, and the present invention contemplates the use ofmixtures of these optical isomers, as well as the individual opticalisomers. Individual optical isomers can be isolated by conventionaloptical resolution techniques.

In the compounds of the invention, R represents an alkyl group havingfrom 1 to 3 carbon atoms, and these groups may be straight or branchedchain groups, specifically the methyl, ethyl, propyl and isopropylgroups. Those compounds of formulae (I), (II) and (III) in which Rrepresents a methyl group are most preferred.

The compounds of the invention may also exist in the form of salts ofthe compounds represented by formulae (I), (II) and (III). The nature ofthe salts is not critical to the invention although, of course, sincethey are intended for therapeutic administration, the salts should bepharmaceutically acceptable salts. Examples of such salts include thealkali and alkaline earth metal salts (such as the sodium or calciumsalts), the aluminium salt, the ammonium salt, salts with organic amines(such as triethylamine, dicyclohexylamine, dibenzylamine, morpholine,piperidine or N-ethylpiperidine) and salts with basic amino acids (suchas lysine or arginine). The salts may be prepared from the freecarboxylic acids of the above formulae by conventional salificationprocesses.

The compounds of the present invention also include the esters ofcompounds of formulae (I), (II) and (III). Examples of such estersinclude C₁ -C₆ alkyl esters, aralkyl esters and pyridylmethyl esters.Examples of alkyl esters include the methyl, ethyl, propyl, isopropyl,butyl, isobutyl, pentyl, isopentyl, hexyl and isohexyl esters; of these,C₁ -C₄ alkyl esters are preferred, particularly the ethyl, methyl,propyl, isopropyl and butyl esters. Examples of aralkyl esters includethe benzyl and phenethyl esters, in which the aromatic ring may besubstituted or unsubstituted. Where it is substituted, the substituentsmay be one or more of the following: C₁ -C₆ groups, e.g. methyl, ethyl,propyl or isopropyl groups; C₁ -C₆ alkoxy groups, e.g. methoxy, ethoxy,propoxy or isopropoxy groups; halogen atoms, e.g. fluorine, chlorine orbromine atoms; or trifluoromethyl groups. In the case of pyridylmethylesters, these may be the 2-, 3- or 4-pyridylmethyl esters.

Compounds of the invention may be prepared by the following methods.

Method A

This is illustrated by the following reaction scheme: ##STR5##

In this, a compound of formula (IV) is reduced to give the compound offormula (I), which may then, if desired, be separated into the cis- andtrans-isomers (II) and (III); alternatively, the cis-isomer can beprepared directly from the compound of formula (IV) by appropriatechoice of reducing agent.

The reduction reaction is preferably carried out in the presence of anorganic solvent, the nature of which is not critical, provided that itdoes not interfere with the reaction. Examples include ethers (such astetrahydrofuran or diethyl ether) and aromatic hydrocarbons (such asbenzene or toluene).

There is also no particular limitation as to the nature of the reducingagent employed, provided that it only reduces the keto group of theketo-carboxylic acid compound of formula (IV). Suitable reducing agentsinclude alkali metal borohydrides, such as sodium borohydride, sodiumcyanoborohydride, potassium tri-sec-butylborohydride and lithiumtri-sec-butylborohydride. The potassium tri-sec-butylborohydride orlithium tri-sec-butylborohydride is preferably employed as thetetrahydrofuran solution thereof sold by Aldrich Chemical Co., Inc.,under the respective registered trade marks K-Selectride orL-Selectride.

When the reducing agent is sodium cyanoborohydride, the reaction ispreferably effected at a pH value of about 3. the reaction temperatureis not particularly critical and the reaction can be effected attemperatures ranging from that achieved by ice-cooling to the refluxtemperature of the solvent employed. The time required for the reactionwill depend upon various factors, mainly the reaction temperature andthe nature of the reducing agent used. It is usually from 10 minutes to3 hours. After completion of the reaction, the compound of formula (I),in the form of a mixture of the cis- and trans-isomers, can be separatedfrom the reaction mixture by conventional means.

The cis- and trans-isomers can be separated from each other byconventional techniques, most especially by high pressure liquidchromatography.

If a tri-sec-butylborohydride such as K-Selectride or L-Selectride isused as the reducing agent, a relatively low temperature is preferablyemployed, e.g. from -78° C. to the temperature of ice-cooling. The timerequired for the reaction will depend mainly upon the reactiontemperature and the nature of the reducing agent, being generally from10 minutes to 5 hours. This reduction selectively gives the cis-isomer(II), which can be separated from the reaction mixture by conventionalmeans after completion of the reaction.

Method B

This is illustrated by the following reaction scheme: ##STR6##

In this reaction, the keto compound of formula (V) is first reduced tothe corresponding hydroxy compound, using the same reagents and underthe same conditions as described in Method A. The resulting hydroxycompound of formula (VI) is then subjected to catalytic reduction, usinghydrogen and a catalyst such as palladium chloride or platinum chloride.This reaction is preferably effected in the presence of a solvent, thenature of which is not critical, provided that it has no adverse effectupon the reaction. Suitable solvents include esters (such as ethylacetate), aromatic hydrocarbons (such as benzene or toluene) andalcohols (such as methanol or ethanol).

After completion of the reaction, the desired compound of formula (I),in the form of a mixture of its cis- and trans-isomers, may be separatedfrom the reaction mixture by conventional means. As in Method A, thecis- and trans-isomers may be separated from each other by conventionaltechniques, especially high pressure liquid chromatography.

The compounds of the invention have been tested for pharmacologicalactivity and found to exhibit anti-inflammatory, analgesic andimmuno-regulatory activities. Details of the pharmacological tests areas follows:

Carrageenin Oedema Test for Anti-inflammatory Activity

Male Wistar rats weighing 120-150 g were fasted overnight and thenreceived a test compound per os as an aqueous tragacanth suspension. 30minutes later, inflammation was induced by the subcutaneous injection of0.05 ml of a 1% w/v carrageenin suspension into the plantar tissue of ahind paw of each rat [Winter et al., Proc. Soc. Exp. Biol. Med., 111,544 (1962)]. The anti-oedema activity was measured volumetrically, byassessing the response, as calculated from the following equation:

    Response=(V-V.sub.o)/V.sub.o

where V_(o) and V represent, respectively, the paw volume immediatelybefore and 3 hours after the carrageenin injection. The test compoundswere administered at various doses and the results are reported in thefollowing Table as the ID₅₀, that is the inhibitory does required toinhibit the response by 50%.

Pain Test for Analgesic Activity

This test was conducted according to a modification of the methodreported by L. O. Randall and J. J. Selitto in Arch. Int. Pharmacodyn.,11, 409 (1959), proposed by Winter and Flatake (1957).

Male Wistar-Imamichi rats of 4 weeks of age and weighing 60-90 g wereinjected with 0.1 ml of a 20% by weight suspension of Brewers' yeast inthe right hind paw. 4 hours later, rats which had a pain threshold topressure-induced pain less than 10×30 g were selected. Each of these wasgiven orally a test compound as an aqueous tragacanth suspension. 1 and2 hours after administration of the test compound, the pain thresholdwas determined by observing pain responses (such as struggling orsqueaking) when the inflamed or normal paw was subjected to pressure bya machine (Ugo-Basile). An "effective" animal was defined, in accordancewith Blane's method (1968), as an animal which showed at least twice themean pain threshold of control animals. The ED₅₀ was calculated by themethod of Litchfield and Wilcoxon (1949).

The compounds tested are identified in the Table as follows:

1=(±)-2-[4-(cis-2-hydroxycyclohexylmethyl)phenyl]-propionic acid;

2=(±)-2-[4-(trans-2-hydroxycyclohexylmethyl)phenyl]-propionic acid

3=Indomethacin;

compounds 1 and 2 being compounds of the invention and compound 3 beinga well known mild analgesic and anti-inflammatory agent.

                  TABLE                                                           ______________________________________                                                    Anti-inflammatory                                                                          Analgesic                                            Compound    activity, ID.sub.50                                                                        activity, ED.sub.50                                  ______________________________________                                        1           3.3 mg/kg    0.86 mg/kg                                           2           0.96 mg/kg   0.76 mg/kg                                           3           2.2 mg/kg     1.6 mg/kg                                           ______________________________________                                    

From the Table, it can be seen that the compounds of the invention haveanalgesic and anti-inflammatory activities comparable with or betterthan the activity of Indomethacin.

Test for Immuno-regulatory Activity

The immuno-regulatory activity of(±)-2-[4-(cis-2-hydroxycyclohexylmethyl)phenyl]propionic acid was testedby Cunningham's method [A. J. Cunningham and A. Szenberg, Immunology 14,599 (1968)]. The test compound was administered orally to female mice ofthe BALB/c strain and simultaneously sheep erythrocytes wereadministered intraperitonially, in order to sensitise the animal tothese erythrocytes as an antigen. After 5 days, the spleen of theexperimental animal was extracted and the number of IgMantibody-producing cells in the spleen cells was calculated. The percentinhibition of antibody production was found to be 50% or more when thedose of compound administered was from 1 to 10 mg per kg.

These results demonstrate that the compounds of the invention havevaluable analgesic, anti-inflammatory and immuno-regulatory activites.

The compounds of the invention are preferably administered in admixturewith a carrier or diluent in the form of a conventional pharmaceuticalcomposition, preferably formulated for oral, rectal or topicaladministration. Compositions for oral administration may be formulatedas, for example, tablets, capsules, granules, powders or syrups,compositions for rectal administration may be in the form ofsuppositories and compositions for topical administration may be in theform of an ointment or a cream. The dosage employed will vary dependingupon the condition, age and body weight of the patient as well as thechosen route of administration, but usually the dose for oraladministration to an adult human being would be from 50 to 300 mg perday, which may be administered in a single dose or in divided doses.

The preparation of the compounds of the invention is further illustratedby the following Examples.

EXAMPLE 1 (±)-2-[4-(2-hydroxycyclohexylmethyl)phenyl]propionic acid

720 mg of (±)-2-[4-(2-oxocyclohexylmethyl)phenyl]-propionic acid weredissolved in 15 ml of tetrahydrofuran, and then 400 mg of sodiumcyanoborohydride were added to the solution. The resulting mixture wasstirred for 40 minutes under ice-cooling, while maintaining the pH at avalue of 3 by the addition of 3N methanolic hydrochloric acid. Ice-waterwas then added to the reaction mixture, which was extracted with diethylether. The extract was dried over anhydrous sodium sulphate and thesolvent was distilled off to give 690 mg of a mixture of the trans- andcis-isomers. This mixture was subjected to high pressure liquidchromatography through silica gel deactivated with acetic acid, using a1:1 by volume mixture of ethyl acetate and hexane as eluent. Thecis-isomer was eluted first, followed by the trans-isomer. Each isomerwas recrystallised from a mixture of diethyl ether and hexane andobtained in the form of crystals.

(±)-2-[4-(trans-2-Hydroxycyclohexylmethyl)phenyl]propionic acid

melting at 117-120 C.

Elemental analysis: Calculated for C₁₆ H₂₂ O₃ : C, 73.25%; H, 8.45%.Found: C, 73.24%; H, 8.40%.

(±)-2-[4-(cis-2-Hydroxycyclohexylmethyl)phenyl]propionic acid

melting at 130°-133° C.

Elemental analysis: Calculated for C₁₆ H₂₂ O₃ : C, 73.25%; H, 8.45%.Found: C, 73.20%; H, 8.43%.

EXAMPLE 2 (±)-2-[4-(2-Hydroxycyclohexylmethyl)phenyl]propionic acid

2.0 g of (+)-2-[4-(2-oxocyclohexylidenemethyl)phenyl]-propionic acid and0.6 g of sodium cyanoborohydride were dissolved in 50 ml of methanol.The pH of the mixture was adjusted to a value of 3 with 6N hydrochloricacid, whilst ice-cooling. The mixture was then stirred, with heating,for 40 minutes. Ice-water was added to the reaction mixture, which wasthen extracted with diethyl ether. The extract was washed with water anddried over anhydrous sodium sulphate, and the solvent was distilled off,to give 350 mg of(±)-2-[4-(2-hydroxycyclohexylidenemethyl)phenyl]propionic acid ascrystals [compound of formula (VI)]. These crystals were dissolved in 10ml of ethyl acetate, 50 mg of palladium chloride were added, and themixture was subjected to catalytic reduction whilst bubbling hydrogenthrough the mixture. After the theoretical amount of hydrogen had beenabsorbed, the catalyst was removed by filtration and the solvent wasdistilled off, to give 330 mg of a mixture of the cis- andtrans-isomers. This mixture was treated in the same manner as in Example1, affording crystals of the cis- and trans-isomers of the titlecompound, having the same properties as the product of Example 1.

EXAMPLE 3 (±)-2-[4-(cis-2-Hydroxycyclohexylmethyl)phenyl]propionic acid

10.0 g of (±)-2-[4-(2-oxocyclohexylmethyl)phenyl]-propionic acid weredissolved in 150 ml of tetrahydrofuran. The solution was cooled to -78°C. and, whilst maintaining the solution at this temperature and under astream of nitrogen, 200 ml of K-Selectride (as a 0.5 molartetrahydrofuran solution) were added dropwise. When the whole of theK-Selectride had been added, the reaction mixture was stirred for 1 hourat 0° C., after which it was cooled to -10° C. and 400 ml of a 0.5Nsolution of hydrochloric acid was added through a dropping funnel. Thereaction mixture was stirred for 1 hour, after which it was extractedwith diethyl ether. The extract was washed with water and dried overanhydrous sodium sulphate. The solvent was distilled off, giving 10.5 gof the title compound in the form of crystals. These were recrystallisedfrom a mixture of diethyl ether and hexane, giving the title compound aspure crystals melting at 130°-133° C.

Elemental analysis: Calculated for C₁₆ H₂₂ O₃ : C, 73.25%; H, 8.45%.Found: C, 73.22%; H, 8.45%.

We claim:
 1. The method of regulating the immune system of a mammal,which comprises administering to said mammal an immuno-regulatory agent,wherein said agent is selected from the group consisting of compounds ofthe formula (I): ##STR7## (wherein R represents hydrogen or a C₁ -C₃alkyl group) and pharmaceutically acceptable salts and esters thereof.2. The method as claimed in claim 1, wherein R represents the methylgroup.
 3. The method as claimed in claim 1, wherein said salt isselected from the group consisting of alkali metal, alkaline earthmetal, aluminium, ammonium, organic base and amino acid salts.
 4. Themethod as claimed in claim 1, wherein said compound is in thecis-configuration with respect to the cyclohexyl ring.
 5. A method ofregulating the immune system of a mammal, which comprises administeringto said mammal an immuno-regulatory agent, wherein said agent isselected from the group consisting of(±)-2-[4-(cis-2-hydroxycyclohexylmethyl)phenyl]propionic acid andpharmaceutically acceptable salts and esters thereof.
 6. The method asclaimed in claim 5 wherein said agent is(±)-2-[4-(cis-2-hydroxy-cyclohexylmethyl)phenyl]propionic acid.